1. Field of the Invention
The current invention concerns Moraxella bovis cytotoxin and a gene encoding Moraxella bovis cytotoxin. In particular, the invention concerns identification, isolation, cloning and identification of nucleotide sequence of the Moraxella bovis gene mbxA and adjacent genes mbxB, mbxC and mbxD, partial purification of the native cytotoxin, preparation of a purified native and recombinant Moraxella bovis cytotoxin, an amino acid sequence of the cytotoxins, preparation of antibodies against the Moraxella bovis cytotoxin, preparation of vaccines against Moraxella bovis and a method for prevention and treatment of infectious bovine keratoconjunctivitis caused by Moraxella bovis. 
2. Background and Related Disclosures
Infectious bovine keratoconjunctivitis (IBK) also called pinkeye, caused by Moraxella bovis (M. bovis) infection ranks as one of the most important cattle diseases in the United States and abroad. Annual estimated incidence of the disease is 5% of all beef cattle with greater than 50% of all herds affected. Epizootics also occur with case attack rates approaching 90-100% of yearling cattle. Affected cattle do not eat and fail to gain weight. Economic losses due to lower market weights of affected cattle and calves and ocular scarring and treatment associated expenses are estimated to exceed $150,000,000 annually. Effective control measures that could substantially reduce this expense are not available as the commercially available vaccines have low efficacy.
Animals affected with IBK exhibit corneal ulceration, edema, ocular pain, photophobia and lacrimation. Control of IBK using antimicrobial treatments has been only partially successful. Antimicrobials have been used to eliminate the carrier state in experimental and field trials, but these treatments have drawbacks that include emergence of resistant bacteria in cattle and the general environment, potential for adulteration of the nation's food supply, high cost and marginal economic benefit.
Topical and oral administration of antibiotics has proven somewhat effective for treatment of pinkeye, but the high cost and losses associated with the ongoing disease often outweigh the benefits of such treatment. Topical therapy alone does not eliminate M. bovis from non-ocular sites such as nasal choanae and vagina where it is known to reside.
Consequently, an effective vaccine or antibody for prevention and treatment of IBK would be an important component of a successful IBK control program.
So far, vaccines prepared from killed or live bacteria, isolated pili, or ribosomes have had limited benefit for immunization of cattle in the field (Proc. Am. Assoc. Bov. Pract., 20:26-32 (1987)). Nevertheless, a large body of field and experimental data exist to show that acquired resistance to pinkeye does develop after the natural occurrence of the infection.
At least two attributes, namely pili and cytotoxin (leukotoxin/hemolysin/cytolysin), of the M. bovis organism are important in the pathogenesis of IBK. M. bovis pili facilitate adhesion of the bacterium to the corneal epithelium and are required for colonization. Pili are highly immunogenic, but have antigenic diversity due to the presence of at least two structural pilin genes and variability in the amino acid composition of the pilin molecule. Variability in pilus gene expression is regulated by a site-specific DNA inversion system. Limited antigenic cross-reactivity was found in vaccines prepared from heterologous pili. M. bovis pili in monovalent or multivalent formulations have demonstrated variable efficacy, attributed to pilus gene inversion that occurs during infection (Vet. Microbiol., 45:129-138 (1995)). The emergence of novel pili serotypes during epizootics of IBK has been documented (Am. J. Vet. Res., 50:1437-1441 (1989)). These studies suggest that because of the pili heterogeneity pilus based vaccines will not be 100% effective at preventing IBK.
The Moraxella bovis cytotoxin (hemolysin) is an important component of immunity to IBK (FEMS Microbiol. Lett., 124:69-74 (1994)).
Moraxella bovis produces a heat labile, approximately 100 kD cytotoxin (hemolysin) that causes cell lysis by forming pores in cell membranes via a calcium-dependent process (Infect. Immunol., 59:1148-1152 (1991)). In vitro, culture filtrates from broth cultures of hemolytic but not nonhemolytic strains of M. bovis cause lysis of bovine neutrophils (Am. J. Vet. Res., 51:191-196 (1990)). In vivo, the ocular damage caused by a purified hemolytic and cytotoxic fraction of M. bovis mimics the lesions seen in naturally occurring IBK (Vet. Microbiol., 42:15-33 (1994)). Unlike the pilin molecule, the M. bovis cytotoxin appears to be more conserved between different M. bovis isolates. Serologic studies show that antihemolytic antibodies recognize hemolysin from different strains of M. bovis (Am. J. Vet. Res., 46:1011-1014 (1985)) and, thus, form an important component of immunity to IBK (FEMS Microbiol. Lett., 124:69-74 (1994)).
In view of the severity of consequences of infectious bovine keratoconjunctivitis, it would be important to have available an effective vaccine and/or anti-Moraxella bovis antibodies that could prevent this condition.
This invention, therefore, concerns an effective IBK vaccine, anti-Moraxella bovis antibodies and to that end an isolation and partial purification of the native Moraxella bovis cytotoxin, a characterization of the M. bovis cytotoxin gene, identification of M. bovis cytotoxin amino acid sequence, preparation of recombinant M. bovis cytotoxin and determination of its efficacy in a vaccine for prophylaxis and treatment of IBK.
All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety.